1Medical Microbiology and Immunology and Rowe Program in Human Genetics, UC Davis School of Medicine, One Shields Avenue,Davis, CA 95616, USA.

Abstract

The widespread use of persistent organic polybrominated diphenyl ethers (PBDEs) as commercial flame retardants has raised concern about potential long-lived effects on human health. Epigenetic mechanisms, such as DNA methylation, are responsive to environmental influences and have long-lasting consequences. Autism spectrum disorders (ASDs) have complex neurodevelopmental origins whereby both genetic and environmental factors are implicated. Rett syndrome is an X-linked ASD caused by mutations in the epigenetic factor methyl-CpG binding protein 2 (MECP2). In this study, an Mecp2 truncation mutant mouse (Mecp2(308)) with social behavioral defects was used to explore the long-lasting effects of PBDE exposure in a genetically and epigenetically susceptible model. Mecp2(308/+) dams were perinatally exposed daily to 2,2',4,4'-tetrabromodiphenyl ether 47 (BDE-47) and bred to wild-type C57BL/6J males, and the offspring of each sex and genotype were examined for developmental, behavioral and epigenetic outcomes. Perinatal BDE-47 exposure negatively impacted fertility of Mecp2(308/+) dams and preweaning weights of females. Global hypomethylation of adult brain DNA was observed specifically in female offspring perinatally exposed to BDE-47 and it coincided with reduced sociability in a genotype-independent manner. A reversing interaction of Mecp2 genotype on BDE-47 exposure was observed in a short-term memory test of social novelty that corresponded to increased Dnmt3a levels specifically in BDE-47-exposed Mecp2(308/+) offspring. In contrast, learning and long-term memory in the Morris water maze was impaired by BDE-47 exposure in female Mecp2(308/+) offspring. These results demonstrate that a genetic and environmental interaction relevant to social and cognitive behaviors shows sexual dimorphism, epigenetic dysregulation, compensatory molecular mechanisms and specific behavioral deficits.

BDE-47, genotype, reversing and compounding effects are observed in behavioral tests, predominantly in females. The chart summarizes significant differences for females versus males of each genotype and treatment independently as well as the Mecp2 by BDE-47 interaction. All behavioral tests described in the Materials and Methods section were performed on all mice for each experiment. Only tests showing significant effects in both Exp. 1 and Exp. 2 are shown.

Sociability and social novelty preference are altered in female mice exposed to BDE-47. (A) Sociability is decreased in both wild-type female and Mecp2308/+ (het) mice with BDE-47 exposure. No genotype effects were seen in the sociability trial in the females, but BDE-47 exposure led to reduced sociability (time in social area minus time in non-social area, F(1,32) = 8.51, P = 0.006). (B) BDE-47 effects on social novelty preference are reversed in Mecp2308/+ het females. An Mecp2 effect and BDE-47 by Mecp2 interaction effect (F(1,31) = 6.12, P = 0.019) were seen for familiar time spent near the now-familiar mouse. BDE-47-exposed het mice spent less time near the familiar stimulus, primarily due to the lower time recorded in the BDE-47-exposed het mice compared with the control het mice. During the social novelty preference trial, no genotype or BDE-47 effects were seen for novelty preference (novel area time minus familiar area time, RMANOVA). (C) The Mecp2 effect for the social novelty preference test is individually graphed as the difference between het and wt mice for percent time, with the familiar mouse to show the significance of the decrease (F(1,31) = 5.52, P = 0.025, asterisk) specifically in BDE-47-exposed mice. (D) The BDE-47 by Mecp2 interaction effect for the social novelty test is individually graphed as the difference between BDE-47- and vehicle-exposed mice for percent time, with the familiar mouse to show the significant reversing interaction (F(1,31) = 6.12, P = 0.019). (E) Social area time in the sociability trial correlated with novel area time in the novelty trial for Mecp2308/+ females treated with BDE-47. Mecp2308/+ mice generally spent less time in the social areas (novel and familiar) than wild-type mice, but this differential was mitigated by BDE-47 treatment. Bar graphs represent least square means and error bars represent SEM. *P < 0.05, **P < 0.02, ***P < 0.01, black represents genotype effects, blue represents BDE-47 effects. Colors of bar graphs for (C) and (D) match those used in the summary table of Figure (blue, decreased; pink, reversing). All data shown are from PND 72 in Exp. 2, but similar interactions effects were observed at PND 40 in Exp. 1. Litter-based statistics were performed on the following group sizes of mice (litters): BDE-47 het n = 9(6), BDE-47 wt n = 8(6), vehicle het n = 12(11), vehicle wt n = 14(12).

BDE-47-induced deficits in learning and memory are compounded by genotype in females. (A) Mean latency in MWM is reduced in BDE-47-treated females. RMANOVA of the latency to reach the platform indicated significant learning across the 4-day period (F(3,33) = 7.96, P < 0.001). A BDE-47 by day interaction (F(3,33) = 3.91, P = 0.017), based on a significant lower latency in the BDE-47 group than the vehicle group observed on the first day. In addition, there was a triple interaction between day, BDE-47 and genotype (F(3,33) = 4.06, P = 0.015). The decrease in latency across days was similar in both vehicle and BDE-47 wild-type females. The decrease in latency across trials was also seen in the vehicle Mecp2308/+ group, but not in the BDE-47 Mecp2308/+ group. (B) The differences in latency shown in (A) for the BDE-47 Mecp2308/+ group were not explained by differences in swim speed. Results from Exp. 1 are shown. Group sizes for mice (litters): BDE-47 het n = 11(6), BDE-47 wt n = 12(7), vehicle het n = 6(4), vehicle wt n = 8(6).

Dnmt3a expression is altered with BDE-47 exposure in Mecp2308/+ but not wild-type females. (A) qRT-PCR analysis of Dnmt1 shows no significant increase between genotype or treatment groups. (B) qRT-PCR analysis of Dnmt3a reveals a significant increase in expression in the het BDE-47 group relative to all other groups. Bar graphs show mean ± SEM, where n = 15 for wt and het vehicle and wt BDE-47 groups, analyzed as three pools of five samples each, and n = 20 for het BDE-47 group, analyzed as four pools of five samples. *P < 0.05, **P < 0.01. Data for males are shown in Supplemental Material, Figure S8.